The main cellular event that occurs during mitosis is the delivery of a complete set of chromosomes to each daughter cell. This is achieved by the attachment of spindle microtubules to kinetochores, specialized structures formed at centromeres. In the earlier grant period, we identified four novel proteins that are kinetochore components. The first examined, CENP-E, has two striking features: its has all of the structural hallmarks of a kinesin-like, microtubule dependent motor and, like mitotic cyclin. It Is quantitatively degraded during the later stages of mitosis. With the direct observation in vivo and in vitro that a kinetochore can translocate laterally along the lattice of a single microtubule, it now seems certain that some, perhaps most or all, aspects of chromosome positioning arc due to kinetochore-bound motors directing chromosome movements. CENP-E is an excellent candidate for such a motor. We now propose to identify how CENP-E binds to kinetochores, to identify and characterize the kinetochore components with which CENP-E interacts, to determine if CENP-E is a motor and, if so, to determine the direction of its movement along microtubules, to examine the mechanism of degradation following the metaphase to anaphase transition, and to use DNA transfection antibody inhibition, antisense methods, and immunodepletion to determine what role(s) CENP-E plays during mitosis. Further, we will exploit antibodies to three other kinetochore components to examine the functional properties of the corresponding proteins, in particular focussing on a 275 kD phosphoprotein whose phosphorylation state changes during mitosis. Lastly, we will also focus on assessing the function of NuMA, an abundant 236 kD nuclear component during interphase, but which binds to the spindle poles during mitosis. Building on our earlier efforts that showed that mutations in NuMA result in post-micronucleation despite normal chromosome segregation, we will now focus more directly on assessing how NuMA participates in the terminal stages of mitotic spindle function (telophase) and/or why NuMA is essential for post- mitotic reassembly of normal full sized nuclei.